Use of genes to increase plant biomass and seed yield

Abstract

The application belongs to the field of plant genetics research, and particularly relates to a gene function for increasing biomass and seed yield of plants. The application discloses a use of a gene for negatively regulating biomass and seed yield of plants, wherein the gene is an AT3G28990 gene with a nucleotide sequence as shown in SEQ ID NO:1, and knocking out the AT3G28990 gene in plants can increase biomass and seed yield of the plants. Knocking out the AT3G28990 gene in Arabidopsis thaliana can effectively improve biomass and yield of the plants, and has a reference significance for high-yield breeding of crops.

Description

Technical Field

[0001] This invention belongs to the field of plant genetics research, specifically involving the function of a gene that increases plant biomass and seed yield. Background Technology

[0002] Plant biomass synthesis is closely related to fruit and seed yield. Increasing the overall biomass of a plant can effectively improve fruit or seed yield, which is of great significance for crop breeding and production practices. Arabidopsis thaliana is a dicotyledonous model plant, and studying the function of genes regulating biomass synthesis in it has important implications for other plants.

[0003] The gene AT3G28990 has been published in The Arabidopsis Information Resource database https: / / www.arabidopsis.org / , but its function and purpose are unknown. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a gene that can effectively increase plant biomass and seed yield, and guide high-yield crop breeding.

[0005] To address the aforementioned technical problems, this invention provides the use of a gene that negatively regulates plant biomass and seed yield. The gene is the AT3G28990 gene with the nucleotide sequence shown in SEQ ID NO: 1. Knocking out the AT3G28990 gene in a plant can increase (significantly increase) the plant's biomass and seed yield.

[0006] An improvement to the use of the gene for negatively regulating plant biomass and seed yield in this invention: the coding region sequence of the AT3G28990 gene is shown in SEQ ID NO:2.

[0007] As a further improvement to the use of the gene for negatively regulating plant biomass and seed yield in this invention: the plant is Arabidopsis thaliana.

[0008] As a further improvement to the use of the gene that negatively regulates plant biomass and seed yield in this invention: the sgRNA sequence of the AT3G28990 gene targeted for knockout: 5'-GAAACCAGTGGACGTGATGA-3'.

[0009] This invention also provides a method for regulating plant biomass and seed yield: knocking out the AT3G28990 gene in the plant to obtain a knockout line of the AT3G28990 gene, the nucleotide sequence of the AT3G28990 gene being shown in SEQ ID NO: 1.

[0010] As an improvement to the method of regulating plant biomass and seed yield of the present invention:

[0011] AT3G28990 gene knockout lines: ko-1, ko-2;

[0012] The nucleotide sequence of ko-1 is shown in SEQ ID NO:4, and the nucleotide sequence of ko-2 is shown in SEQ ID NO:6.

[0013] The present invention relates to the gene AT3G28990 of the model plant Arabidopsis thaliana, the nucleotide sequence of which is shown in SEQ ID NO:1, and the protein sequence which is encoded is shown in SEQ ID NO:2.

[0014] This invention also provides a method for knocking out the AT3G28990 gene in Arabidopsis thaliana, comprising the following steps:

[0015] 1) Using CRISPR / Cas9 technology, design the sgRNA sequence for targeted knockout of the AT3G28990 gene: 5'-GAAACCAGTGGACGTGATGA-3';

[0016] 2) Use the sequence from step 1) to synthesize primers and construct a CRISPR / Cas9 vector;

[0017] 3) Genetically transform the vector obtained in step 2) into the wild-type Arabidopsis thaliana variety Col-0 to obtain the corresponding transgenic plants; from the transgenic plants, two different mutation types of the AT3G28990 gene, the lines ko-1 and ko-2, were identified.

[0018] The specific technical solution of the present invention is as follows:

[0019] Using CRISPR / Cas9 gene editing technology, a specific sgRNA sequence targeting the AT3G28990 gene was designed based on its nucleotide sequence (SEQ ID NO:1) at its coding region. A corresponding CRISPR / Cas9 vector was constructed and genetically transformed into the wild-type Arabidopsis thaliana variety Col-0 to obtain transgenic plants. PCR amplification and sequencing of the AT3G28990 gene in the transgenic plants identified two non-mutant lines of the AT3G28990 gene: ko-1 (1 base insertion) and ko-2 (28 base loss + 6 base insertion). Figure 1All of these mutations resulted in a frameshift mutation in the AT3G28990 gene, prematurely terminating protein translation, effectively knocking out the gene. In the ko-1 mutant line, the coding sequence of the AT3G28990 gene is SEQ ID NO:4, and the protein coding sequence is SEQ ID NO:5; in the ko-2 mutant line, the coding sequence of the AT3G28990 gene is SEQ ID NO:6, and the protein coding sequence is SEQ ID NO:7.

[0020] Compared with the wild-type Arabidopsis control, the fresh weight of mutant lines ko-1 and ko-2 was significantly higher. Figure 1 ), plant dry weight ( Figure 2 ), number of fruits per plant ( Figure 3 ), weight of seeds ( Figure 4 The significant increase in both values ​​indicates that knocking out the AT3G28990 gene in Arabidopsis thaliana can effectively improve plant biomass and yield, which has reference value for high-yield crop breeding. Attached Figure Description

[0021] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0022] Figure 1 The image shows the editing status of the AT3G28990 gene target site in Arabidopsis thaliana ko-1 and ko-2 mutant plants. Arrows or underlines indicate the location of mutations.

[0023] Figure 2 The fresh weight of the aboveground parts of Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type controls was determined.

[0024] Figure 3 The plant dry weight of the aboveground parts of Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type controls was determined.

[0025] Figure 4 The number of siliques per plant in Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type controls was statistically analyzed.

[0026] Figure 5 The thousand-seed weight of Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type controls was determined.

[0027] Figures 2-5 The values ​​in the table are mean ± standard deviation. ** indicates that there is a highly significant difference between the ko-1 or ko-2 mutant plants and the wild-type control (P < 0.01) according to the t-test analysis. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0029] Example 1: Construction of the Arabidopsis AT3G28990 gene knockout vector

[0030] Based on the AT3G28990 gene sequence (SEQ ID NO:1), a targeted editing sgRNA sequence was designed at its coding region: 5'-GAAACCAGTGGACGTGATGA-3'. The AT3G28990 gene knockout vector was constructed using a CRISPR / Cas9 kit (Biogle, China), following the product instructions.

[0031] Example 2: Genetic transformation of Arabidopsis thaliana using the AT3G28990 gene knockout vector

[0032] The CRISPR / Cas9 vector constructed in Example 1 was genetically transformed into the wild-type Arabidopsis thaliana variety Col-0. The transformation method was based on the literature (Plant Journal, 1998, 16(6):735-743), and the corresponding transgenic Arabidopsis thaliana plants (including ko-1 and ko-2) were obtained.

[0033] Example 3: Identification of Arabidopsis thaliana AT3G28990 gene knockout plants

[0034] Take 0.1 g of fresh leaves from the wild-type Arabidopsis thaliana variety Col-0 and transgenic plants, grind them with liquid nitrogen, add 300 μL of extraction buffer (0.1 mol / L Tris-HCl pH 8.0, 500 mmol / L NaCl, 1.25 g / L SDS), incubate at 65℃ for 1 h, shaking 2-3 times during incubation, add 100 μL of 5 mol / L KAC, shake well, incubate on ice for 10 min, add 250 μL of chloroform, mix well, let stand for 5 min, centrifuge at 8000 r / min for 10 min, transfer 250 μL of supernatant to a new 1.5 ml tube, add 250 μL of pre-chilled isopropanol, shake well until flocculent precipitate appears, refrigerate for 10 min, centrifuge at 12000 r / min, 4℃ for 5 min, discard the supernatant, add 1 mL of 70% ethanol, centrifuge at 12000 r / min for 7 min, discard the supernatant, air dry upside down at room temperature, add 80 μL of... Mix thoroughly with ddH2O and store at -20℃ for later use.

[0035] Primers for PCR amplification of the AT3G28990 gene were synthesized: upstream 5'-CATGTGCATGCGTTTCTTCT-3', downstream 5'-GCTTCTGCTCGTACGTCTCA-3'. Using DNA from the wild-type Arabidopsis thaliana Col-0 and transgenic plants as templates, PCR amplification was performed using 2×Taq PCR reagent (Tiangen, Beijing). The PCR amplification system consisted of: 1 μL template DNA, 1 μL 2×Taq PCRMasterMixⅡ, 1 μL primers (upstream + downstream mixture, 10 μM), and ddH2O to a final volume of 20 μL. The PCR amplification program was: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ extension for 2 min.

[0036] The coding region sequence of the AT3G28990 gene in WT plants is shown in SEQ ID NO:2, encoding an 88-amino acid protein, the sequence of which is shown in SEQ ID NO:3. Sequencing analysis of the PCR products from the transgenic plants identified two mutant types of the AT3G28990 gene:

[0037] In ko-1 plants, one base was inserted into the AT3G28990 gene. Figure 1 This mutation causes premature termination of the gene's protein translation, resulting in a protein encoding only 53 amino acids. The mutated coding sequence is SEQ ID NO:4, and the encoded protein sequence is SEQ ID NO:5.

[0038] In ko-2 plants, 28 bases were lost and 6 bases were inserted into the AT3G28990 gene. Figure 1 This mutation causes the translation of the gene to terminate prematurely, encoding only 44 amino acids. The mutated coding sequence is SEQ ID NO:6, and the encoded protein sequence is SEQ ID NO:7.

[0039] Example 4: Biomass statistics of Arabidopsis thaliana AT3G28990 gene knockout plants

[0040] On the same day of maturity, 10 wild-type, 10 ko-1, and 10 ko-2 plants of Arabidopsis thaliana AT3G28990 gene were randomly selected. After removing the roots, the fresh weight of the aboveground parts of each plant was measured. Then, the plants were dried at 80℃, and the dry weight of the aboveground parts of each plant was measured. The t-test method was used to analyze the significant differences.

[0041] The results are as follows Figure 2 and Figure 3As shown, the fresh weight and dry weight of the aboveground parts of AT3G28990 gene knockout ko-1 and ko-2 plants were significantly higher than those of the wild-type control, indicating that knocking out this gene can effectively increase the biomass synthesis of Arabidopsis plants.

[0042] Example 5: Determination of 1000-grain weight of Arabidopsis thaliana

[0043] On the same day of maturity, 10 Arabidopsis thaliana AT3G28990 gene knockout ko-1 and ko-2 plants and 10 wild-type controls were randomly selected, and the number of siliques on the main stem of each plant was counted. Then, all siliques of each plant were removed, and the seeds of each plant were collected. The weight of 1000 seeds per plant was weighed using an electronic balance. The t-test method was used to analyze the significance of differences.

[0044] The results are as follows Figure 4 and Figure 5 As shown, the number of siliques and the thousand-seed weight of AT3G28990 gene knockout ko-1 and ko-2 plants were significantly higher than those of the wild-type control. This indicates that knocking out this gene can effectively increase the number of fruits and seed weight in Arabidopsis thaliana, thereby improving seed yield.

[0045] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The use of genes that negatively regulate plant biomass and seed yield, characterized by: The gene is the AT3G28990 gene with the nucleotide sequence shown in SEQ ID NO:

1. Knocking out the AT3G28990 gene in plants can increase plant biomass and seed yield; the plant is Arabidopsis thaliana.

2. The use of the gene for negatively regulating plant biomass and seed yield according to claim 1, characterized in that: The coding region sequence of the AT3G28990 gene is shown in SEQ ID NO:

2.

3. The use of the gene for negatively regulating plant biomass and seed yield according to claim 1 or 2, characterized in that: The sgRNA sequence for targeted knockout of the AT3G28990 gene is: 5'- GAAACCAGTGGACGTGATGA-3'.

4. A method for regulating plant biomass and seed yield, characterized in that: The AT3G28990 gene was knocked out in a plant to obtain a knockout line of the AT3G28990 gene. The nucleotide sequence of the AT3G28990 gene is shown in SEQ ID NO:

1. The plant is Arabidopsis thaliana.

5. The method for regulating plant biomass and seed yield according to claim 4, characterized in that: Knockout lines of the AT3G28990 gene: ko-1 and ko-2; the nucleotide sequence of ko-1 is shown in SEQ ID NO: 4, and the nucleotide sequence of ko-2 is shown in SEQ ID NO: 6.

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